Fast green fcf

Spider mite adult females were collected and fixed overnight at 4°C in 4% formaldehyde in 10 mM phosphate buffer saline (PBS), pH 7.4 with 1% (v/v) Triton X-100. Mites were washed twice in 10 mM PBS and were subsequently dehydrated in an ethanol series (10%, 30%; 50% and 70%; v/v in H₂O). Further dehydration and paraffin embedding were performed in tissue processor (Leica ASP300TP). Embedded mites were sectioned on a microtome (Leica RM2255 Microtome) at a thickness of 5 μm. Sections were dewaxed in two 10 min changes of 100% xylene and were progressively rehydrated. The following histological dyes were used as general staining: 0.1% Safranin O (C.I. 50240; MilliporeSigma) and 0.05% Fast Green (FCF, C.I. 42053, MilliporeSigma), 1% hematoxylin (C.I. 75290, MilliporeSigma) solution and 1% eosin (C.I. 45400, MilliporeSigma), and Movats' pentachrome solution. Images were obtained using a Zeiss AxioCam Color HRc CCD Camera 412-312 and Zeiss Axioplan II microscope. Finally, 300 nM of DAPI (D1306, Invitrogen) mixed in 10 mM PBS was used to visualize nuclei under epifluorescence Zeiss Axioplan II microscope.

Fabricated cell sheets were fixed with 4% paraformaldehyde for 30 min at RT. Harvested rat knee tissue was fixed in 4% paraformaldehyde for four days and decalcified in RapidCal Immuno (BBC Biochemical, Mount Vernon, USA) for one day at RT. Samples were embedded in paraffin blocks and then cut into 5-µm transverse sections with a microtome. Slides were deparaffinized by baking in an oven at 65 °C and subsequent washes with xylene and ethanol. Sections were hydrated by gradual ethanol replacement by distilled water. Safranin-O was used for metachromatic staining for sulfated glycosaminoglycans. Samples were stained for 5 min with Wiegert’s Iron Hematoxylin (MilliporeSigma), 5 min with 0.5 g/L Fast Green FCF (MilliporeSigma), and 5 min with 0.1% Safranin-O (MilliporeSigma). Images were taken with a BX41 microscope (Olympus, Tokyo, Japan) and AmScope Software (USA).